This is the U.S. National Phase under 35 U.S.C. xc2xa7371 of International Application PCT/KR00/01236, filed Oct. 30, 2000, which clams priority of Korean Application No. 1999/47379, filed Oct. 29, 1999.
1. Field of the Invention
The present invention relates to a novel nystatin-resistant microorganism belonging to Aspergillus genus and a process for preparing triol heptanoic acid, a precursor of 3-hydroxy-3-methylglutaryl-coenzyme A (xe2x80x9cHMG-CoAxe2x80x9d) reductase inhibitor by employing the said microorganism, more specifically, to a novel nystatin-resistant mutant which provides a high productivity of triol heptanoic acid with minimal productivity of analogues therefrom, produced by introducing mutations into a protoplast fusion mutant strain Aspergillus terreus CLS216-7 producing mevinolinic acid with a high yield and a process for preparing triol hedtanoic acid by aerobic submerged culture of the novel mutant strain in a nutrient medium.
2. Description of the Prior Art
Hyperlipidemia is known as one of the major factors for development of cardiovascular disease, the leading cause of death. The substances developed for treatment of hyperlipidemia are mostly inhibitors of HMG-CoA reductase involved in the cholesterol biosynthesis, and are useful for treatment of hypercholesterolemia and hyperlipidemia in human body.
Lovastatin, developed first and now commercially available as a therapeutic agent against hyperlipidemia, is produced by fermentation of fungi and specific microorganisms, for example, Aspergillus terreus (see: Korean patent publication No. 83-2438; U.S. Pat. No. 4,231,936) or Monascus genus (see: Korean patent publication No. 83-2329). Lovastatin is also a starting material for the synthesis of simvastatin, a potent inhibitor of HMG-CoA reductase (see: U.S. Pat. No. 5,223,415).
Triol heptanoic acid, chemically represented as 7-[1,2,6,7,8,8a(R)-hexahydro-2(S), 6(R)-dimethyl-8(S)-hydroxy-1(S)-naphthyl]-3(R),5(R)-dihyroxyheptanoic acid, serves as a starting material for the synthesis of simvastatin or other 8-ester analogues which function as potent HMG-CoA reductase inhibitors (see: Korean patent publication No. 85-669). Triol heptanoic acid can be produced by chemical hydrolysis of lovastatin, fermentation of Monascus ruber (see: Japanese patent laid-open publication No. 86-13798; Endo et al., The Journal of Antibiotics, 38(3):420-422, 1985), or fermentation of mutated Aspergillus terreus MF-4833, a mevinolinic acid-producing microorganism (see: U.S. Pat. No. 5,250,435).
Mevinolinic acid, produced by Aspergillus terreus, is biosynthesis via polyketide pathway in a similar manner to the fatty acid and sterol biosynthesis (see: Moore et al., J. Am. Chem. Soc., 107:3694-3701, 1985), and it has 2 methyl groups transferred from S-adenosyl methionine (SAM) (see: Shiao, M. S. and Pon, H. S., Proc. Nat. Sci. Counc. B. Roc., 11:223-231, 1987). Recently, two polyketide-synthesizing enzymes engaged in forming a backbone consisting of 9 acetate units and genes thereof were isolated from Aspergillus terreus. Analysis of the genes encoding an enzyme synthesizing 2-methylbutyrate side chain of mevinolinic acid and an enzyme adding the side chain to triol heptanoic acid, backbone of mevinolinic acid, revealed that their deduced amino acid sequences and presumed active sites are almost similar to those of a fatty acid synthetase (see: WO95/12661; Kennedy, J. et al., Science, 284:1368-1372, 1999).
In mutant strains of Aspergillus terreus and yeasts resistant to nystatin, a kind of polyenic macrolide antibiotics, which affects biosynthesis of fatty acids and sterols and cell membrane permeability, the nystatin makes some changes in fatty acid and sterol compositions of cell membrane (see: Mazumder, C. et al., Lipids, 22(9):609-612, 1987) and also affects the biosynthesis of secondary metabolites in the mutants (see: Liu, Y. T., Proc. Natl. Sci. Counc. Repub. China [B], 8(2):182-186, 1984).
Meanwhile, by mutating a disclosed strain of mevinolinic acid-producing Aspergillus terreus using nitrosoguanidine, it has been reported that the triol heptanoic acid was produced up to 5.95 g/L and the product on of mevinolinic acid was markedly reduced in a 14 day-fermentation process (see: European patent publication No. 0517413A1).
However, it has revealed a shortcoming that the fermentation process is time consuming and produces analogues of triol heptanoic acid with a high level, which naturally limits the application of fermentation process for producing simvastatin or 8-ester analogues, in industrial scale. In this regard, the chemical hydrolysis of lovastatin has prevailed in the art, since it is rather advantageous for the production of triol heptanoic acid. The production of triol heptanoic acid from lovastatin by chemical hydrolysis is, however, proven to be less satisfactory in a sense that the cost is very high.
Accordingly, there are strong reasons for exploring and developing a novel microbe which produces triol heptanoic acid with a high yield and has relatively short fermentation time to reduce cost and production period.
The present inventors isolated a novel mutant strain Aspergillus terreus CLS247-13 resistant to nystatin, an inhibitor of fatty acid and sterol biosynthesis, by treating Aspergillus terreus strain producing mevinolinic acid with conventional mutagens known in the art, followed by selecting a mutant strain producing triol heptanoic acid with a high yield while producing only a little triol heptanoic acid analogues during a short period of fermentation and maintaining its genetic characteristics stably through many generations.
A primary object of present invention is,, therefore, to provide a novel microorganism belonging to Aspergillus genus, which shows resistance to nystatin and produces triol heptanoic acid with a high yield.
The other object of the invention is to provide a process for preparing triol heptanoic acid employing the said microorganism.